Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
The process of bone formation (osteogenesis) involves three main steps: (i) production of the extracellular organic matrix (osteoid); (ii) mineralization of the matrix to form bone; and (iii) bone remodeling by resorption and reformation. The cellular activities of osteoblasts, osteocytes, and osteoclasts are essential to the process. Osteoblasts synthesize the collagenous precursors of bone matrix and also regulate its mineralization. As the process of bone formation progresses, the osteoblasts come to lie in tiny spaces (lacunae) within the surrounding mineralized matrix and are then called osteocytes. The cell processes of osteocytes occupy minute canals (canaliculi) which permit the circulation of tissue fluids. To meet the requirements of skeletal growth and mechanical function, bone undergoes dynamic remodeling by a coupled process of bone resorption by osteoclasts and reformation by osteoblasts.
Osteoblasts are cells of mesenchymal origin. They possess a single nucleus, have a shape that varies from flat to plump, reflecting their level of cellular activity, and in later stages of maturity line up along bone-forming surfaces. Osteoblasts synthesize and lay down precursors of collagen, which comprises 90-95% of the organic matrix of bone. Osteoblasts also produce the proteoglycans of ground substance and are rich in alkaline phosphatase, an organic phosphate-splitting enzyme. Osteoblasts have receptors for parathyroid hormone and possibly also for estrogen. Hormones, growth factors, physical activity, and other stimuli act through osteoblasts to bring about their effects on bone.
The collagen formed by osteoblasts is typically deposited in parallel or concentric layers to produce mature (lamellar) bone. But when bone is rapidly formed, as in the fetus or certain pathological conditions (fracture callus, fibrous dysplasia, hyperparathyroidism), the collagen is not deposited in a parallel array but in a basket-like weave and is called woven, immature, or primitive bone. In fully decalcified bone sections, the extracellular matrix stains pink with H+E, similar to collagen elsewhere but with a more homogeneous than fibrillar structure which latter is easily observed by polarizing microscopy.
The main mineral component of bone is an imperfectly crystalline hydroxyapatite [Ca10(PO4)6(OH)21 which comprises about ¼ the volume and ½ the mass of normal adult bone. The mineral crystals, as shown by electron microscopy, are deposited along, and in close relation to, the bone collagen fibrils. Calcium and phosphorus (Pi) are, of course, derived from the blood plasma and ultimately from nutritional sources. Vitamin D metabolites and parathormone (PTH) are important mediators of calcium regulation, and lack of the former or excess of the latter leads to bone mineral depletion.
The extracellular matrix of bone is mineralized soon after its deposition, but a very thin layer of unmineralized matrix is seen on the bone surface, and this is called the osteoid layer or osteoid seam. In some pathological conditions, the thickness and extent of the osteoid layer may be increased (hyperosteoidosis) or decreased. Hyperosteoidosis may be caused by conditions of delayed bone mineralization (as in osteomalacia/rickets resulting from vitamin D deficiency) or of increased bone formation (as in fracture callus, Paget's disease of bone, etc.).
Osteoclasts are multinucleated cells, apparently of monocyte-phagocyte origin, which adhere to the surface of bone undergoing resorption and lie in depressions termed Howship's lacunae or resorption bays. With remodeling, the resorptive surfaces become covered by osteoblasts which form new bone at the site. Several metabolic bone diseases (such as hyperparathyroidism, Paget's disease, and others) are characterized by increased modeling and increased osteoclastic activity. Osteoclasts are apparently activated by “signals” from osteoblasts. For example, osteoblasts have receptors for PTH whereas osteoclasts do not, and PTH-induced osteoclastic bone resorption is said not to occur in the absence of osteoblasts.
At an early stage of human embryonic development, a cartilage model of much of the skeleton (of extremities, trunk, and base of the skull) is formed from the mesenchyme. In the further fetal development of long bones, a rim of primitive bone is first laid down in layers over the middle of the shaft by osteoblasts arising from the overlying periosteum, and subperiosteal bone formed in this way soon extends up and down the shaft (diaphysis). The process by which bone tissue replaces membranous fibrous tissue is called intramembranous ossification. This is the process by which the diaphysis increases in width throughout postnatal growth. Some bones, such as the flat bones of the calvarium, are formed entirely, or in great part, by intramembranous ossification.
The cartilage cells of the core of the fetal shaft degenerate upon contact with penetrating buds of periosteal osteoblasts, the cartilage matrix becomes mineralized and resorbed, and the resulting surfaces and spaces are lined by osteoblasts which lay down woven bone and form primitive bone trabeculae. The process by which bone tissue replaces cartilage is called endochondral ossification and begins in the femur at about the ninth week of fetal life. Some of the trabeculae fuse with the subperiosteal new bone while others are resorbed to form a medullary cavity which will be occupied by hematopoietic tissue. Thus, the primitive bone shaft is formed and lies between the cartilaginous ends which become the epiphyses. In the later months of fetal life, the woven bone of the diaphysis will be replaced by lamellar bone of mature type. Over time, the bone cortex is thickened and remodeled to serve mechanical functions and is permeated by haversian systems (bone-forming units) of longitudinal, vascularized canals bounded by concentric lamellae of bone, culminating in the typical appearance of compact cortical bone as seen in the adult.
The longitudinal growth of the long bones occurs in the epiphysial (epiphyseal) growth plate as cartilage cells, arising from reserve cells, undergo mitosis and proliferate in orderly longitudinal columns. The proliferated cartilage cells vacuolate as they move toward the cartilage-bone junction (metaphysis), the cartilage matrix becomes mineralized, and buds of osteoblasts emerging from the metaphysis replace the mineralized cartilage with bone. This process of cartilage cell proliferation and endochondral ossification is repeated over and over, and the bones become longer and larger. Meanwhile, at age-related intervals, secondary centers of endochondral ossification (“radiologic epiphysis”) begin to form on the articular side of the growth plate.
When skeletal maturity is reached, the cartilage cells of the growth plate cease to proliferate, the growth plate becomes thinner, is replaced by bone and disappears, and the epiphysis is “closed” or fused with the shaft.
The recent identification of the genetic basis of hereditary skeletal disorders, such as dwarfism and osteochondrodysplasias, is providing important insights into the intricate processes of skeletal formation, growth and homeostasis. These processes include patterning events during condensation and differentiation of mesenchymal cells to form cartilage precursors of the future bones, the replacement of cartilage by bones through endochondral ossification, the growth of long bones through proliferation and differentiation of chondrocytes in growth plates, and bone formation through differentiation of osteoblasts from mesenchymal cells in areas of intramembranous ossification. Defects in any of these processes can give rise to skeletal abnormalities. Mutations in transcription factors such as HOX and PAX and members of the TGFβ superfamily cause disorders associated with abnormal mesenchymal condensation, while defects in the transcription factor SOX-9 lead to abnormalities in chondrocyte differentiation.